Dataset Information

Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis.

ABSTRACT: AzoA from Enterococcus faecalis is a member of the polymeric flavin-dependent NADH-preferred azoreductase group. Little is known about the binding and interaction of NADH and azo dye in the azoreductase group. A synergetic strategy based on computational prediction, reverse genetics validation coupled with site-directed mutagenesis, and reconstruction of mutation network was used to investigate the binding and interaction of NADH and a model azo dye, Methyl Red, with AzoA. Methyl Red and NADH interacted in a unique binding mode in which the benzoic acid moiety of Methyl Red and the nicotinamide ring of NADH were not parallel to the flavin isoalloxazine ring, but lay against it at angles of ?45° and ?35°, respectively. The adenine ribose moiety of NADH was surrounded by loop ?2 on chain B and ?3 on chain A in a typical Rossmann fold. There were 12 and 19 amino acid residues that could participate in the binding of Methyl Red and NADH, respectively, especially the residues Tyr-129 and Asp-184. The functional perturbation effects of 13 residues, including Tyr-129 and Asp-184, were mapped to reconstruct the mutation network, which confirmed the proposed binding modes and also provided insights into the interaction among NADH, FMN and Methyl Red.

SUBMITTER: Feng J

PROVIDER: S-EPMC5862418 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis.

Feng Jinhui J Kweon Ohgew O Xu Haiyan H Cerniglia Carl E CE Chen Huizhong H

Archives of biochemistry and biophysics 20120224 2

AzoA from Enterococcus faecalis is a member of the polymeric flavin-dependent NADH-preferred azoreductase group. Little is known about the binding and interaction of NADH and azo dye in the azoreductase group. A synergetic strategy based on computational prediction, reverse genetics validation coupled with site-directed mutagenesis, and reconstruction of mutation network was used to investigate the binding and interaction of NADH and a model azo dye, Methyl Red, with AzoA. Methyl Red and NADH in ...[more]

PMID: 22387379

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Project description:The emergence of antimicrobial-resistant and virulent enterococci is a major public health concern. While enterococci are commonly found in food of animal origin, the knowledge on their zoonotic potential is limited. The aim of this study was to determine and compare the antimicrobial susceptibility and virulence traits of Enterococcus faecalis and Enterococcus faecium isolates from human clinical specimens and retail red meat in Slovenia. A total of 242 isolates were investigated: 101 from humans (71 E. faecalis, 30 E. faecium) and 141 from fresh beef and pork (120 E. faecalis, 21 E. faecium). The susceptibility to 12 antimicrobials was tested using a broth microdilution method, and the presence of seven common virulence genes was investigated using PCR. In both species, the distribution of several resistance phenotypes and virulence genes was disparate for isolates of different origin. All isolates were susceptible to daptomycin, linezolid, teicoplanin, and vancomycin. In both species, the susceptibility to antimicrobials was strongly associated with a food origin and the multidrug resistance, observed in 29.6% of E. faecalis and 73.3% E. faecium clinical isolates, with a clinical origin (Fisher's exact test). Among meat isolates, in total 66.0% of E. faecalis and E. faecium isolates were susceptible to all antimicrobials tested and 32.6% were resistant to either one or two antimicrobials. In E. faecalis, several virulence genes were significantly associated with a clinical origin; the most common (31.0%) gene pattern included all the tested genes except hyl. In meat isolates, the virulence genes were detected in E. faecalis only and the most common pattern included ace, efaA, and gelE (32.5%), of which gelE showed a statistically significant association with a clinical origin. These results emphasize the importance of E. faecalis in red meat as a reservoir of virulence genes involved in its persistence and human infections with reported severe outcomes.

Project description:UnlabelledThough the bacterial opportunist Enterococcus faecalis causes a myriad of hospital-acquired infections (HAIs), including catheter-associated urinary tract infections (CAUTIs), little is known about the virulence mechanisms that it employs. However, the endocarditis- and biofilm-associated pilus (Ebp), a member of the sortase-assembled pilus family, was shown to play a role in a mouse model of E. faecalis ascending UTI. The Ebp pilus comprises the major EbpC shaft subunit and the EbpA and EbpB minor subunits. We investigated the biogenesis and function of Ebp pili in an experimental model of CAUTI using a panel of chromosomal pilin deletion mutants. A nonpiliated pilus knockout mutant (EbpABC(-) strain) was severely attenuated compared to its isogenic parent OG1RF in experimental CAUTI. In contrast, a nonpiliated ebpC deletion mutant (EbpC(-) strain) behaved similarly to OG1RF in vivo because it expressed EbpA and EbpB. Deletion of the minor pilin gene ebpA or ebpB perturbed pilus biogenesis and led to defects in experimental CAUTI. We discovered that the function of Ebp pili in vivo depended on a predicted metal ion-dependent adhesion site (MIDAS) motif in EbpA's von Willebrand factor A domain, a common protein domain among the tip subunits of sortase-assembled pili. Thus, this study identified the Ebp pilus as a virulence factor in E. faecalis CAUTI and also defined the molecular basis of this function, critical knowledge for the rational development of targeted therapeutics.ImportanceCatheter-associated urinary tract infections (CAUTIs), one of the most common hospital-acquired infections (HAIs), present considerable treatment challenges for physicians. Inherently resistant to several classes of antibiotics and with a propensity to acquire vancomycin resistance, enterococci are particularly worrisome etiologic agents of CAUTI. A detailed understanding of the molecular basis of Enterococcus faecalis pathogenesis in CAUTI is necessary for the development of preventative and therapeutic strategies. Our results elucidated the importance of the E. faecalis Ebp pilus and its subunits for enterococcal virulence in a mouse model of CAUTI. We further showed that the metal ion-dependent adhesion site (MIDAS) motif in EbpA is necessary for Ebp function in vivo. As this motif occurs in other sortase-assembled pili, our results have implications for the molecular basis of virulence not only in E. faecalis CAUTI but also in additional infections caused by enterococci and other Gram-positive pathogens.

Dataset Information

Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis.

Publications

Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis.

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